Neuropeptide Y receptor Y5 and nucleic acid sequences

ABSTRACT

The present invention provides novel NPY/PYY receptor proteins and the nucleic acid sequence encoding them. The invention is directed to the isolation, characterization, and pharmacological use of these receptors and nucleic acids. In particular, this invention provides human and rat NPY/PYY receptors (which we call the NPY Y5 receptor) and nucleic acids. Also provided are recombinant expression constructs useful for transfecting cells and expressing the protein in vitro and in vivo. The invention further provides methods for detecting expression levels of the protein as well as methods for screening for receptor antagonists and agonists to be used for the treatment of obesity or anorexia, respectively.

BACKGROUND OF THE INVENTION

This is a continuation-in-part application of U.S. Provisional Ser. No.60/014,969 filed on Apr. 8, 1996.

1. Field of the Invention

This invention relates to a novel neurotransmitter Neuropeptide Yreceptor, its nucleic acid sequence, and compounds, compositions, andmethods for their use.

2. Summary of the Related Art

Neuropeptide Y (NPY) is a 36-amino acid peptide neurotransmitter that islocated throughout the central and peripheral nervous systems. Tatemoto,Proc. Natl. Acad Sci. USA 79, 5485 (1982); Hazlewood, Proc. Soc. Exp.Biol. Med. 202, 44 (1993). It affects a broad range of phenomena,including blood pressure regulation, memory, anxiolysis/sedation, foodand water appetite, vascular and other smooth muscle activity,intestinal electrolyte secretion, and urinary sodium excretion. E.g.,Colners and Wahlestedt, The Biology of Neuropeptide Y and RelatedPeptides (Humana Press, Totowa, N.J., 1993); Kalra et al., Phys. &Behavior 50, 5 (1991).

Peptide YY (PYY) is also a 36 amino acid peptide and has significantsequence homology (70%) to NPY. Tatemoto et al., Nature 296, 659 (1982).Its anatomical distribution is similar to that of NPY, although it islocated mainly in the endocrine cells of the lower gastrointestinaltract. Bottcher et al., Regul. Pept. 8, 261 (1984). Like NPY, PYYstimulates feeding in rats. Morley et al., Brain Res. 341, 200 (1985).Along with the pancreatic polypeptide (PP), NPY and PYY have a commontertiary structure, characterized by the so-called PP-fold. Glover, Eur.J. Biochem. 142, 379 (1985). Both NPY and PYY show about a 50% sequencehomology with PP.

Because of their structural similarities, NPY and PYY have a number ofcommon receptors. At least four receptor subtypes, Y1, Y2, Y3, andY4/PP, have been identified. The affinity for NPY, PYY, and variousfragments thereof varies among the subtypes. See, e.g., Bard et al. (WO95/17906) and references cited therein. For example, Y1 and Y2 subtypeshave high affinity for NPY and PYY. Whereas Y1 has high affinity for(Leu³¹Pro³⁴)NPY ((LP)NPY) and low affinity for (13-36)NPY, Y2 behavesoppositely. Y3 has high affinity for NPY but low affinity for PYY. Y4/PPhas a high affinity for PP but relatively low affinity for NPY.

Wahlestedt (WO 93/24515) and Larhammar et al. (J. Biol. Chem. 267, 10935(1992)) describe the cloning and identification of the human Y1-typeNPY/PYY receptor isolated from human fetal brain tissue. Selbie et al.(WO 93/09227) disclosed the full length cDNA sequence of the Y1 receptorfrom human hippocampus. Eva et al. (FEBS Lett. 271, 81 (1990)) clonedthe NPY Y1 receptor from rat forebrain. Eva et al. (FEBS Lett. 314, 285(1992)) cloned the NPY Y1 receptor from murine genomic DNA.

The Y2-type receptor has also been cloned. Gerald et al. (WO 95/21245)disclosed the cDNA sequence of human hippocampal Y2 and two rat Y2clones. Rose et al. (J. Biol. Chem. 270, 22661 (1995)) disclosed thecDNA sequence of the Y2 receptor from a human neuroblastoma cell line.

Bard et al. (supra) and Lundell et al. (J. Biol. Chem. 270, 29123(1995)) described cloning the cDNA sequence of the Y4/PP receptor fromboth rat spleen and human placenta.

To date, the Y3 receptor has not been cloned.

Because of the important role of NPY and PYY in a number ofphysiological processes, such as feeding, there is a strong need tofurther develop materials and methods for investigating the mechanisticbehavior of these compounds and for treating diseased and other abnormalstates associated with the physiological processes in which NPY and PYYact. Specifically, the NPY analogs/fragments that induce feeding, suchas (LP)(3-36)NPY, do not bind to the previously identified NPY/PYYreceptors with affinities consistent with the feeding response.Accordingly, there is a need and desire to identify the NPY/PYY receptorthat is responsible for the feeding response. Antagonists to such areceptor could be used to treat obesity and diabetes by reducingappetite and food consumption.

SUMMARY OF THE INVENTION

The present invention provides, inter alia, novel NPY/PYY receptorproteins. Also provided are the nucleic acid sequences encoding thesenovel receptor proteins, as well as compounds and methods for usingthese proteins and their nucleic acid sequences.

The present invention provides novel proteins, nucleic acids, andmethods useful for developing and identifying compounds for thetreatment of such diseases and disorders as obesity. Identified anddisclosed herein is the protein sequence for a novel receptor for theneurotransmitters Neuropeptide Y (NPY) and Peptide YY (PYY) and thenucleic acid sequence encoding this receptor, which we call the NPY Y5(or simply “Y5”) receptor. The importance of this discovery ismanifested in the effects of NPY, which include blood pressureregulation, memory enhancement, anxiolysis/sedation, and increased foodintake. Thus, this receptor protein is useful for screening for NPY/PYYagonist and antagonist activity for controlling these conditions.

In one aspect of the present invention, we provide isolated nucleic acidsequences for a novel NPY and PYY receptor, the Y5 receptor. Inparticular, we provide the cDNA sequences encoding for the rat and humanreceptors and isoforms thereof. These nucleic acid sequences have avariety of uses. For example, they are useful for making vectors and fortransforming cells, both of which are ultimately useful for productionof the Y5 receptor protein. They are also useful as scientific researchtools for developing nucleic acid probes for determining receptorexpression levels, e.g., to identify diseased or otherwise abnormalstates. They are useful for developing analytical tools such asantisense oligonucleotides for selectively inhibiting expression of thereceptor gene to determine physiological responses.

In another aspect of the present invention, we provide a homogenouscomposition comprising the receptor Y5 protein. The protein is usefulfor screening drugs for agonist and antagonist activity, and, therefore,for screening for drugs useful in regulating physiological responsesassociated with the Y5 receptor. Specifically, antagonists to the Y5receptor could be used to treat obesity and diabetes by reducingappetite and food consumption, whereas agonists could be used for thetreatment of anorexic conditions. The proteins are also useful fordeveloping antibodies for detection of the protein.

Flowing from the foregoing are a number of other aspects of theinvention, including (a) vectors, such as plasmids, comprising thereceptor Y5 nucleic acid sequence that may further comprise additionalregulatory elements, e.g., promoters, (b) transformed cells that expressthe Y5 receptor, (c) nucleic acid probes, (d) antisenseoligonucleotides, (e) agonists, (f) antagonists, and (g) transgenicmammals. Further aspects of the invention comprise methods for makingand using the foregoing compounds and compositions.

The foregoing merely summarizes certain aspects of the present inventionand is not intended, nor should it be construed, to limit the inventionin any manner. All patents and other publications recited herein arehereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays the competition curves of various peptides for [¹²⁵I]PYYto Y5 receptor membranes transiently expressed in COS-7 cells.

FIG. 2 displays saturation curves for specific binding of [¹²⁵I]PYY toY5 receptor membranes transiently expressed in COS-7 cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention comprises, in part, a novel NPY/PYY receptorprotein, the Y5 receptor. Particularly preferred embodiments of the Y5receptor are those having an amino acid sequence substantially the sameas SEQ ID NOs 2, 4, or 6. As used herein, reference to the Y5 receptoris meant as a reference to any protein having an amino acid sequencesubstantially the same as SEQ ID NOs 2, 4, or 6. The present inventionalso comprises the nucleic acid sequence encoding the Y5 protein, whichnucleic acid sequences is substantially the same as SEQ ID NOs 1, 3, or5. Receptors SEQ ID NOs 2 and SEQ ID NO 4 are rat Y5 receptors andappear to be allelic variations, with SEQ ID NO 4 the most commonlyoccurring and, therefore, the preferred embodiment of the rat Y5receptor of this invention. SEQ ID NO 6 is the human Y5 receptor and itspreferred embodiment.

As used herein, a protein “having an amino acid sequence substantiallythe same as SEQ ID NO x” (where “x” is the number of one of the proteinsequences recited in the Sequence Listing) means a protein whose aminoacid sequence is the same as SEQ ID NO x or differs only in a way suchthat IC₅₀[(3-36)NPY], IC₅₀[(Leu³¹Pro³⁴)NPY], andIC₅₀[(Leu³¹Pro³⁴)(3-36)NPY] as determined according to the methoddetailed in Example 4, infra, are less than or equal to 30 nM. The NPYfragments (3-36)NPY, (Leu³¹Pro³⁴)NPY and (Leu³¹Pro³⁴)(3-36)NPY induce afeeding response. Those skilled in the art will appreciate thatconservative substitutions of amino acids can be made withoutsignificantly diminishing the protein's affinity for NPY, PYY, andfragments and analogs thereof. Other substitutions may be made thatincrease the protein's affinity for these compounds. Making andidentifying such proteins is a routine matter given the teachingsherein, and can be accomplished, for example, by altering the nucleicacid sequence encoding the protein (as disclosed herein), inserting itinto a vector, transforming a cell, expressing the nucleic acidsequence, and measuring the binding affinity of the resulting protein,all as taught herein.

As used herein the term “a molecule having a nucleotide sequencesubstantially the same as SEQ ID NO y” (wherein “y” is the number of oneof the protein-encoding nucleotide sequences listed in the SequenceListing) means a nucleic acid encoding a protein “having an amino acidsequence substantially the same as SEQ ID NO y+1” (wherein “y+1” is thenumber of the amino acid sequence for which nucleotide sequence “y”codes) as defined above. This definition is intended to encompassnatural allelic variations in the Y5 sequence. Cloned nucleic acidprovided by the present invention may encode Y5 protein of any speciesof origin, including (but not limited to), for example, mouse, rat,rabbit, cat, dog, primate, and human. Preferably the nucleic acidprovided by the invention encodes Y5 receptors of mammalian, and mostpreferably, rat or human origin.

The invention also includes nucleotide sequences encoding chimericproteins comprised of parts of the Y5 receptor and parts of otherrelated seven-transmembrane receptors.

The 6B clone (SEQ ID NO 1) (see Example 2, infra) has a 2.4 kb cDNAinsert with a open reading frame from nucleotide 248 to 1582 thatencodes a 445 amino acid protein (SEQ ID NO 2). Hydrophobicity plotanalysis using PEPPLOT of GCG shows that the Y5 receptor has seventransmembrane-like domains, indicating it might be a G-protein-coupledreceptor. Unlike other known subtypes of NPY receptor family, the thirdintracellular loop of the Y5 receptor is unusually long. Another novelfeature of the Y5 peptide sequence is that it has a much shorterC-terminal tail sequence than other known members of the NPY receptorfamily. It is also important to note that the Y5 sequence shows only30-33% amino acid sequence identity to other NPY receptors.

Nucleic acid hybridization probes provided by the invention are DNAsconsisting essentially of the nucleotide sequences complementary to anysequence depicted in SEQ ID NOs 1, 3, and 5 that is effective in nucleicacid hybridization. Nucleic acid probes are useful for detecting Y5 geneexpression in cells and tissues using techniques well-known in the art,including, but not limited to, Northern blot hybridization, in situhybridization, and Southern hybridization to reversetranscriptase—polymerase chain reaction product DNAs. The probesprovided by the present invention, including oligonucleotide probesderived therefrom, are also useful for Southern hybridization ofmammalian, preferably human, genomic DNA for screening for restrictionfragment length polymorphism (RFLP) associated with certain geneticdisorders. As used herein, the term complementary means a nucleic acidhaving a sequence that is sufficiently complementary in the Watson-Cricksense to a target nucleic acid to bind to the target under physiologicalconditions or experimental conditions those skilled in the art routinelyuse when employing probes.

Receptor Y5 binds various fragments and analogs of NPY and PYY withaffinities different from that of the known receptors. The rank order ofbinding affinity of receptor Y5 was found to be:NPY=(LP)NPY=PYY=(3-36)NPY=(LP)(3-36)NPY>(10-36)NPY>(18-36)NPYTable 1, infra, presents a more detailed affinity profile of the Y5receptor for NPY, PYY, and various fragments thereof. As used herein, aprotein having substantially the same affinity profile as the Y5receptor means a protein in which the IC₅₀ of each of the peptideslisted in Table 1, infra, is no more than an order of magnitude greaterthan those listed in Table 1 for each of the respective peptides asmeasured according to the methods described in Example 4. Importantly,the NPY analogs/fragments that induce feeding, such as (LP)(3-36)NPY, donot bind to the previously identified NPY/PYY receptors with affinitiesconsistent with the feeding response.

The production of proteins such as receptor Y5 from cloned genes bygenetic engineering means is well known in this art. The discussionwhich follows is accordingly intended as an overview of this field, andis not intended to reflect the full state of the art.

DNA which encodes receptor Y5 may be obtained, in view of the instantdisclosure, by chemical synthesis, by screening reverse transcripts ofmRNA from appropriate cells or cell line cultures, by screening genomiclibraries from appropriate cells, or by combinations of theseprocedures, as illustrated below. Screening of mRNA or genomic DNA maybe carried out with oligonucleotide probes generated from the Y5 genesequence information provided herein. Probes may be labeled with adetectable group such as a fluorescent group, a radioactive atom or achemiluminescent group in accordance with known procedures and used inconventional hybridization assays, as described in greater detail in theExamples below. In the alternative, the Y5 gene sequence may be obtainedby use of the polymerase chain reaction (PCR) procedure, with the PCRoligonucleotide primers being produced from the Y5 gene sequenceprovided herein. See U.S. Pat. Nos. 4,683,195 to Mullis et al. and4,683,202 to Mullis.

Receptor Y5 may be synthesized in host cells transformed with arecombinant expression construct comprising a nucleic acid encoding thereceptor Y5. Such a recombinant expression construct can also becomprised of a vector that is a replicable DNA construct. Vectors areused herein either to amplify DNA encoding Y5 and/or to express DNAwhich encodes Y5. For the purposes of this invention, a recombinantexpression construct is a replicable DNA construct in which a DNAsequence encoding Y5 is operably linked to suitable control sequencescapable of effecting the expression of Y5 in a suitable host. The needfor such control sequences will vary depending upon the host selectedand the transformation method chosen. Generally, control sequencesinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences which control the termination oftranscription and translation. Amplification vectors do not requireexpression control domains. All that is needed is the ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants. See,Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition,Cold Spring Harbor Press, New York, 1989).

Vectors useful for practicing the present invention include plasmids,viruses (including phage), retroviruses, and integratable DNA fragments(i.e., fragments integratable into the host genome by homologousrecombination). The vector replicates and functions independently of thehost genome, or may, in some instances, integrate into the genomeitself. Suitable vectors will contain replicon and control sequenceswhich are derived from species compatible with the intended expressionhost. The vectors may be self-replicating. Suitable vectors for thepurposes of the present invention include pBluescript, pcDNA3, and, forinsect cells, baculovirus. A preferred vector is the plasmid pcDNA3(Invitrogen).

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniquesthat are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and relegated inthe form desired.

Site-specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions that are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. See,e.g., New England Biolabs, Product Catalog. In general, about 1 μg ofplasmid or DNA sequence is cleaved by one unit of enzyme in about 20 μlof buffer solution. Often excess of restriction enzyme is used to ensurecomplete digestion of the DNA substrate. Incubation times of about onehour to two hours at about 37° C. are workable, although variations aretolerable. After each incubation, protein is removed by extraction withphenol/chloroform, and may be followed by ether extraction. The nucleicacid may be recovered from aqueous fractions by precipitation withethanol. If desired, size separation of the cleaved fragments may beperformed by polyacrylamide gel or agarose gel electrophoresis usingstandard techniques. A general description of size separations is foundin Methods in Enzymology 65, 499-560 (1980).

Transformed host cells are cells which have been transformed ortransfected with recombinant expression constructs made usingrecombinant DNA techniques and comprising mammalian Y5-encodingsequences. Preferred host cells for transient transfection are COS-7cells. Transformed host cells may ordinarily express Y5, but host cellstransformed for purposes of cloning or amplifying nucleic acidhybridization probe DNA need not express the receptor. When expressed,the mammalian Y5 protein will typically be located in the host cellmembrane. See, Sambrook et al., ibid.

Cultures of cells derived from multicellular organisms are desirablehosts for recombinant Y5 protein synthesis. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. However, mammalian cells are preferred, asillustrated in the Examples. Propagation of such cells in cell culturehas become a routine procedure. See Tissue Culture (Academic Press,Kruse & Patterson, Eds., 1973). Examples of useful host cell lines arebacteria cells, insect cells, yeast cells, human 293 cells, VERO andHeLa cells, LMTK cells, and W1138, BHK, COS-7, CV, and MDCK cell lines.Human 293 cells are preferred.

The invention provides homogeneous compositions of mammalian Y5 producedby transformed eukaryotic cells as provided herein. Such homogeneouscompositions are intended to be comprised of mammalian Y5 protein thatcomprises at least 90% of the protein in such homogenous composition.The invention also provides membrane preparation from cells expressingY5 as the result of transformation with a recombinant expressionconstruct, as described here.

Mammalian Y5 protein made from cloned genes in accordance with thepresent invention may be used for screening compounds for Y5 agonist orantagonist activity, or for determining the amount of a Y5 agonist orantagonist drug in a solution (e.g., blood plasma or serum). Forexample, host cells may be transformed with a recombinant expressionconstruct of the present invention, Y5 protein expressed in those hostcells, the cells lysed, and the membranes from those cells used toscreen compounds for Y5 binding activity. Competitive binding assays inwhich such procedures may be carried out are well known in the art. Byselection of host cells which do not ordinarily express Y5, pure orcrude preparations of membranes containing Y5 can be obtained. Further,Y5 agonists and antagonists can be identified by transforming host cellswith a recombinant expression construct as provided by the presentinvention. Membranes obtained from such cells (and membranes of intactcells) can be used in binding studies wherein the drug dissociationactivity is monitored.

It is known that the neurotransmitter NPY is a regulator of appetite. Asshown herein, the various NPY analogs/fragments that induce feeding,such as (LP)(3-36)NPY, bind with a high affinity to the Y5 receptor.Conversely, the NPY analogs/fragments that bind to the Y5 receptor witha lower affinity, such as (20-36)NPY, do not elicit feeding. It istherefore evident that by contacting the Y5 receptor with agonists andantagonists, feeding can be modulated. Accordingly, antagonists to theY5 receptor, identified by the methods described herein, can be used toreduce appetite and hence treat obesity, diabetes and hyperlipidemia,and, conversely, agonists to the Y5 receptor can be used to treatconditions such as anorexia

This invention provides a pharmaceutical composition comprising aneffective amount of a agonist or antagonist drug identified by themethod described herein and a pharmaceutically acceptable carrier. Suchdrugs and carrier can be administered by various routes, for exampleoral, subcutaneous, intramuscular, intravenous or intracerebral. Thepreferred route of administration would be oral at daily doses of about0.01-100 mg/kg.

This invention provides a method of treating obesity, diabetes orhyperlipidemia, wherein the abnormality is improved by reducing theactivity of Y5 receptor or blocking the binding of ligands to a Y5receptor, which method comprises administering an effective amount ofthe antagonist-containing pharmaceutical composition described above tosuppress the subject's appetite. Similarly, the invention also providesmethods for treating diseases and conditions resulting from underfeedingand/or a loss of appetite, which method comprises administering aneffective amount of an agonist-containing pharmaceutical compositiondescribed above to stimulate the subject's appetite.

The recombinant expression constructs of the present invention areuseful in molecular biology to transform cells which do not ordinarilyexpress Y5 to thereafter express this receptor. Such cells are useful asintermediates for making cell membrane preparations useful for receptorbinding assays, which are in turn useful for drug screening. Drugsidentified from such receptor assays can be used for the treatment ofobesity, diabetes or anorexia.

The recombinant expression constructs of the present invention are alsouseful in gene therapy. Cloned genes of the present invention, orfragments thereof, may also be used in gene therapy carried out byhomologous recombination or site-directed mutagenesis. See generallyThomas & Capecchi, Cell 51, 503-512 (1987); Bertling, Bioscience Reports7, 107-112 (1987); Smithies et al., Nature 317, 230-234 (1985).

Oligonucleotides of the present invention are useful as diagnostic toolsfor probing Y5 gene expression in tissues. For example, tissues areprobed in situ with oligonucleotide probes carrying detectable groups byconventional autoradiographic techniques, as explained in greater detailin the Examples below, to investigate native expression of this receptoror pathological conditions relating thereto. Further, chromosomes can beprobed to investigate the presence or absence of the Y5 gene, andpotential pathological conditions related thereto, as also illustratedby the Examples below. Probes according to the invention shouldgenerally be at least about 15 nucleotides in length to prevent bindingto random sequences, but, under the appropriate circumstances may besmaller.

The invention also provides antibodies that are immunologically reactiveto a mammalian Y5, preferably rat or human Y5. The antibodies providedby the invention are raised in animals by inoculation with cells thatexpress a mammalian Y5 or epitopes thereof, using methods well known inthe art. Animals that are used for such inoculations include individualsfrom species comprising cows, sheep, pigs, mice, rats, rabbits,hamsters, goats and primates. Preferred animals for inoculation arerodents (including mice, rats, hamsters) and rabbits. The most preferredanimal is the mouse.

Cells that can be used for such inoculations, or for any of the othermeans used in the invention, include any cell line which naturallyexpresses a mammalian Y5, or any cell or cell line that expresses amammalian Y5 or any epitope thereof as a result of molecular or geneticengineering, or that has been treated to increase the expression of amammalian Y5 by physical, biochemical or genetic means. Preferred cellsare human cells, most preferably HEK 293 and BHK cells that have beentransformed with a recombinant expression construct comprising a nucleicacid encoding a mammalian Y5, preferably a rat or human Y5, and thatexpress the mammalian Y5 gene product.

The present invention provides monoclonal antibodies that areimmunologically reactive with an epitope of mammalian Y5 or fragmentthereof and that is present on the surface of mammalian cells,preferably human or mouse cells. These antibodies are made using methodsand techniques well known to those of skill in the art.

Monoclonal antibodies provided by the present invention are produced byhybridoma cell lines, that are also provided by the invention and thatare made by methods well known in the art. Hybridoma cell lines are madeby fusing individual cells of a myeloma cell line with spleen cellsderived from animals immunized with cells expressing the Y5 receptor,preferably rat or human cells, as described above. The myeloma celllines used in the invention include lines derived from myelomas of mice,rats, hamsters, primates and humans. Preferred myeloma cell lines arefrom mouse. The animals from whom spleens are obtained afterimmunization are rats, mice and hamsters, preferably mice, mostpreferably Balb/c mice. Spleen cells and myeloma cells are fused using anumber of methods well known in the art, including but not limited toincubation with inactivated Sendai virus and incubation in the presenceof polyethylene glycol (PEG). The most preferred method for cell fusionis incubation in the presence of a solution of 45% (w/v) PEG-1450.Monoclonal antibodies produced by hybridoma cell lines can be harvestedfrom cell culture supernatant fluids from in vitro cell growth;alternatively, hybridoma cells can be injected subcutaneously and/orinto the peritoneal cavity of an animal, most preferably a mouse, andthe monoclonal antibodies obtained from blood and/or ascites fluid.

Monoclonal antibodies provided by the present invention are alsoproduced by recombinant genetic methods well known to those of skill inthe art, and the present invention encompasses antibodies made by suchmethods that are immunologically reactive with an epitope of a mammalianY5.

The present invention encompasses fragments of the antibody that areimmunologically reactive with an epitope of a mammalian Y5. Suchfragments are produced by any number of methods, including but notlimited to proteolytic cleavage, chemical synthesis or preparation ofsuch fragments by means of genetic engineering technology. The presentinvention also encompasses single-chain antibodies that areimmunologically reactive with an epitope of a mammalian Y5 made bymethods known to those of skill in the art.

The present invention also encompasses an epitope of a mammalian Y5 thatis comprised of sequences and/or a conformation of sequences present inthe mammalian Y5 molecule. This epitope may be naturally occurring, ormay be the result of proteolytic cleavage of the mammalian Y5 moleculeand isolation of an epitope-containing peptide or may be obtained bysynthesis of an epitope-containing peptide using methods well known tothose skilled in the art. The present invention also encompasses epitopepeptides produced as a result of genetic engineering technology andsynthesized by genetically engineered prokaryotic or eukaryotic cells.

The invention also includes chimeric antibodies, comprised of lightchain and heavy chain peptides immunologically reactive to an epitopethat is a mammalian Y5. The chimeric antibodies embodied in the presentinvention include those that are derived from naturally occurringantibodies as well as chimeric antibodies made by means of geneticengineering technology well known to those of skill in the art.

Also provided by the present invention are non-human transgenic animalsgrown from germ cells transformed with the Y5 nucleic acid sequenceaccording to the invention and that express the Y5 receptor according tothe invention and offspring and descendants thereof. Also provided aretransgenic non-human mammals comprising a homologous recombinationknockout of the native Y5 receptor, as well as transgenic non-humanmammals grown from germ cells transformed with nucleic acid antisense tothe Y5 nucleic acid of the invention and offspring and descendantsthereof. Further included as part of the present invention aretransgenic animals which the native Y5 receptor has been replaced withthe human homolog. Of course, offspring and descendants of all of theforegoing transgenic animals are also encompassed by the invention.

Transgenic animals according to the invention can be made using wellknown techniques with the nucleic acids disclosed herein. E.g., Leder etal., U.S. Pat. Nos. 4,736,866 and 5,175,383; Hogan et al., Manipulatingthe Mouse Embryo, A Laboratory Manual (Cold Spring Harbor Laboratory(1986)); Capecchi, Science 244, 1288 (1989); Zimmer and Gruss, Nature338, 150 (1989); Kuhn et al., Science 269, 1427 (1995); Katsuki et al.,Science 241, 593 (1988); Hasty et al., Nature 350, 243 (1991); Stacey etal., Mol. Cell Biol. 14, 1009 (1994); Hanks et al., Science 269, 679(1995); and Marx, Science 269, 636 (1995). Such transgenic animals areuseful for screening for and determining the physiological effects of Y5receptor agonists and antagonist. Consequently, such transgenic animalsare useful for developing drugs to regulate physiological activities inwhich NPY and/or PYY participate.

The following Examples are provided for illustrative purposes only andare not intended, nor should they be construed, as limiting theinvention in any manner.

EXAMPLES Example 1 Isolation and Sequencing of Rat Y5 Receptor

Isolation of Rat Hypothalamus mRNA and Construction of cDNA Library

Expression cloning strategy was used to clone novel NPY receptor in rathypothalamus cDNA library. RNA was obtained from 9 frozen rathypothalami weighing a total of 0.87 grams. Poly(A) RNA was isolateddirectly from the tissue using the Promega PolyATtract System 1000 kit(Promega, Madison, Wis.). The hypothalami were homogenized in 4 mL of 4Mguanidine thiocyanate-25 mM sodium citrate, pH 7.1-2% β-mercaptoethanolusing a Polytron at full-speed for approximately 1 minute. To thehomogenized tissue 8 mL of 4M guanidine thiocyanate-25 mM sodiumcitrate, pH 7.1-1% β-mercaptoethanol which had been preheated to 70° C.was added. After mixing thoroughly, 870 pmol biotinylated oligo(dT) wasadded; the mixture was incubated at 70° C. for 5 minutes. The homogenatewas subjected to centrifugation at 12000×g for 10 minutes at roomtemperature; the homogenate was transferred to a clean tube and 10.44 mLStreptavidin MAGNESPHERE® Paramagnetic Particles (SA-PMPs) which hadbeen prepared as per the published protocol was added. (Promega Corp.published protocol TM 228; Promega Corporation, Madison, Wis.). Thehomogenate and SA-PMPs were incubated together for 2 minutes at roomtemperature after which the homogenate was decanted while theSA-PMP-biotinylated oligo(dT)-hypothalamic poly(A) RNA complex wasretained in the tube by a magnetic stand. The complex was washed as perthe protocol, after which the RNA was precipitated and resuspended inwater. 25 micrograms of this poly(A) RNA was used by Invitrogen(Invitrogen Corporation, San Diego, Calif.) to prepare a cDNA expressionlibrary. The protocols used by Invitrogen to prepare the cDNA libraryare essentially based upon the procedures of Okayama and Berg (Molec.Cell Biol. 2, 161 (1982)) and Gubler and Hoffman (Gene 25, 263 (1983))(Invitrogen Corporation publications 130813sa and 130928sa). Anoligo(dt) anchor primer was used for reverse transcription, and thelibrary was cloned unidirectionally into pcDNA3 vector which contains aCMV promoter for eukaryotic expression. The cDNA library had 5.3×10⁵primary recombinants with an average insert size of 2.59 kb.

Isolation of a Novel Y5 Receptor cDNA Clone

The rat hypothalamus cDNA library was plated on the LB/Ampicillin platesin pools of 1,000 independent colonies. The plates were incubated at 37°C. for about 20 hours and the bacteria from each plate were scraped in4-5 ml LB/Ampicillin media. Two ml of the bacteria samples were used forplasmid preparation and one ml of each pool was stored at −80° C. in 15%glycerol.

COS-7 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, GIBCO11965-092), 10% fetal bovine serum (GIBCO 16000-028), and 1×antibiotic/antimycotic solution (GIBCO 15240-039) (Gaithersburg, Md.).Cells were trypsinized and split at 50 to 70% confluency.

DNA from 1300 pools was transfected into COS-7 cells for [¹²⁵I]PYYbinding assays. Twenty four hours before transfection, cells were platedinto flaskette chambers (Nunc, Inc. 177453, Naperville, Ill.) at 3×10⁵cells/flaskette (equivalent to 3×10⁴ cells/cm²). Two μg of plasmid DNAfrom each pool was transfected into the cells using 10 μl ofLipofectamine (GIBCO 18324-012) according to the manufacture's protocol.Forty eight hours after transfection, the [¹²⁵I]PYY binding assay wasperformed in the flaskette chamber. The cells were treated with 2 mltotal binding buffer: 10 mM HEPES, 5 mM KCl, 1.2 mM KH₂PO₄, 2.5 mMCaCl₂, 1.2 mM MgSO₄, 150 mM NaCl, 25 mM NaHCO₃, 10 mg/ml bovine serumalbumin, 0.5 mg/ml bacitracin and 0.4 mg/ml soybean trypsin inhibitor atroom temperature for 15 minutes. The cells were then incubated with 100pM porcine [¹²⁵I]PYY (Amersham (Arlington Heights, Ill.), SpecificActivity 4000 Ci/mmol) in the total binding buffer for 90 minutes atroom temperature. After binding, the cells were washed three times withice-cold total binding buffer without ligand and one time with coldphosphate buffered saline (PBS). Cells were fixed with 1% coldglutaraldehyde in PBS for 15 minutes, washed once with cold PBS/0.5 MTris, pH 7.5 and incubated in PBS/0.5 M Tris, pH 7.5 for 15 minutes at4° C. After washing one more time with cold PBS, the slides were dippedin 0.5% gelatin at 42° C. and dried under vacuum. The dried slides weredipped in 50% photographic emulsion (Kodak (Rochester, N.Y.) NTB2) at42° C. and exposed in the darkbox for four days at 4° C. After four daysof exposure, the darkbox was moved to room temperature for one hour andslides were developed in developer D-19 (Kodak) for three minutes at 15°C. and fixed in fixer (Kodak) for three minutes at 15° C., washed inwater and air dried. Cells were stained with Diff-Quik stain set(Baxter, McGaw Park, Ill.) and air dried. Slides were dipped intoxylenes and mounted with DPX mountant (Electron Microscopy Science, FortWashington, Pa.). Positive cells were identified using dark fieldmicroscopy.

Twenty one positive pools were identified. Since the hypothalamusexpresses different subtypes of NPY receptors including Y1 and Y2receptors, we analyzed all the positive pools for Y1, Y2 and Y4/PPreceptors by PCR. Of the 21 positive pools tested as described above, 12pools contained Y1, 4 pools contained Y2 and none contained Y4/PP. Fivepools (Y217, Y555, Y589, Y861 and Y1139) were negative by PCR analysis.The pool Y217 was subdivided in 24 subpools of 200 colonies, then 50colonies, and finally a single clone, the Y217.24.13.6B clone (6B), wasisolated.

DNA and Peptide Sequences Analysis

Plasmid DNA was sequenced by Lark Technologies Inc. (Houston, Tex.) andBiotechnology Resource Laboratory of Yale University (New Haven, Conn.)using Sequenase Kit (US Biochemical, Cleveland, Ohio) or AppliedBiosystems' automatic sequencer system (model 373A). The peptidesequence was deduced from the long open-reading-frame of the nucleotidesequence. DNA and peptide sequences were analyzed using the GCG program(Genetics Computer Group, Madison, Wis.). The results are embodied inSEQ ID NO 1 (the nucleic acid sequence) and SEQ ID NO 2 (the amino acidsequence).

Example 2 Localization of Rat Y5 Receptor in Brain and Other Tissues

Northern Blot

To study the expression level of the Y5 receptor in the rat brain andother tissues, we did Northern blot analysis using the 6B 2.4 kb probe.A rat multiple tissue Northern blot (Clontech Laboratories, Palo Alto,Calif.) was hybridized to the ³²P-labeled rat 6B probe. The blotcontains 2 μg of poly A⁺ RNA per lane from rat heart, brain, spleen,lung, liver, skeletal muscle, kidney, and testis. Hybridization wascarried out in 1× hybridization solution containing 6×SSC (0.9 M NaCl,0.09 M Na Citrate, pH 7.0), 5× Denhardt's solution (0.1%polyvinylpyrrolidone, 0.1% ficoll type 400, 0.1% bovine serum albumin),100 mg/ml sheared, and denatured salmon sperm DNA at 65° C. The filterwas washed at 65° C. in 0.1×SSC, 0.1% SDS and exposed to Kodak XAR 5film with two intensifying screens. A single 2.6 kb band was detected inthe brain after overnight exposure of the blot. No bands were found fromother tissues (heart, spleen, lung, liver, skeletal muscle, kidney andtestis) in the Clontech multiple tissue Northern blot, even after sixdays of exposure.

We tested 6B expression in more rat tissues and different regions ofbrain. mRNA was isolated from rat whole brain, cortex, hypothalamus,hippocampus, olfactory bulb, spleen, stomach, kidney, small intestine,adrenal and pancreas using Fast Track Isolation Kit (Invitrogen). Ten μgof mRNA from different brain regions and multiple tissues were run on adenaturing formaldehyde 1% agarose gel, transferred to a Nytran membrane(Schleicher and Schuell) and hybridized with ³²P-labeled 6B 2.4 kb probeand washed at high stringency. After overnight hybridization, the filterwas washed at high stringency and exposed to X-ray film withintensifying screens. The 6B receptor mRNA was detectable in the brainregions examined after one day exposure, but no signal was observed fromother tissues, even after a week exposure with double intensifyingscreens.

Example 3 Isolation of Two Isoforms of the Rat Y5 Receptor

Plasmid DNA from pools Y555, Y589, and Y861 described in Example 1 werehybridized to the Y5 probe at high stringency. A single positive clonewas isolated from the Y555 pool and sequenced as described in Example 1.Compared to the 6B DNA sequence, the Y555 sequence (SEQ ID NO 4 has a123 bp insert sequence located at the 5′-untranslated region betweennucleotides 239 and 240 of Y5 clone. The coding region of the clonesY555, Y589, and Y861 has the same sequence as clone 6B, except for onenucleotide substitution (C to T) at position 430 of the 6B clone. Thenucleotide substitution changes the amino acid proline to leucine in thefirst transmembrane domain. The corresponding amino acid sequence isgiven by SEQ ID NO 4.

The different isoforms of the receptor could be the allelic variants ofthe same gene. To test this hypothesis, we analyzed genomic DNA from 16rats. The genomic DNA from each animal was used as template for PCRanalysis. A 314 bp DNA fragment that contains the site of the nucleotidevariation was amplified and sequenced. Of the 16 DNA samples tested, 14samples had a T at position 430 and 2 samples had a C. This resultstrongly suggests that the amino acid variation is an allelic variant.

Example 4 Pharmacological Characterization of the Novel Rat NPYReceptors

Transient Transfection

Monkey kidney cells (COS-7) were maintained in T-175 cm² flasks (NUNC)at 37° C. with 5% CO₂ in a humidified atmosphere. Cells were grown inDulbecco's Modified Eagle Medium (DMEM) supplemented with 2 mMglutamine, 10% fetal bovine serum, 1 mM sodium pyruvate, andantibiotic/antimycotic. Cells at 70% confluency were transfected with Y5DNA using the Lipofectamine method (GIBCO-BRL). 15 μg DNA and 90 μlLipofectamine were added to each flask. Media was completely replaced 24hours post transfection, and membranes were harvested 24 hours later.

Stable Expression of the Rat NPY Y5 receptor (Clone Y861)

A strain of the human embryonic kidney cell line 293 adapted to grow insuspension (293S) was used for these experiments. Approximately 1×10⁶cells were seeded onto a 10-cm dish 24 hours prior to transfection. Therat NPY Y5 cDNA (Y861), subcloned in the eukaryotic expression vectorpcDNA3 (Invitrogen, Carlsbad, Calif.) was first linearized with NotI andpurified using a Wizard PCR Prep kit (Promega). In preparation fortransfection, 15 μg of the linearized DNA were added to 500 μl of DMEMcell culture media, and 30 μl of Lipofectamine (Life Sciences) wereadded into a separate 500 μl aliquot of DMEM. These two solutions weremixed together and incubated for 20 minutes at room temperature and theresulting DNA/lipid complexes were then slowly added to the cells (whichhad been previously rinsed once with serum-free DMEM) and covered with atotal volume of 10 ml. Cells were then transferred to a humidified 10%CO₂ incubator and left for 4 hours at 37° C., at which time the mediawas replaced with DMEM supplemented with 8% FBS. After 16 hours, cellswere trypsinized and split at a 1:15 ratio into 10-cm dishes containingDMEM/8% FBS in the presence of 700 μg/ml of G418 (selection media). Whendiscrete colonies became apparent (after approximately 10 days), cellswere pooled and carried through 2 additional passages in selectionmedia. Cells were then trypsinized and diluted in preparation forcloning by limited dilution (CBLD), such that an average of one cell wasseeded in each well of a 96-well microtiter culture plate, and wasinspected periodically for the subsequent 2 to 3 weeks. After 21 days inculture under selection conditions, those wells containing singlecolonies were selected and transferred to 24-well culture platesfollowing trypsinization. Each of these clones was propagated untilsufficient quantities were available for testing [¹²⁵I]PYY bindingactivity, from which one particular clone designated E7 was selected onthe basis of its high level of binding activity.

Stable Expression of the Human NPY Y5 Receptor

293 cells were plated onto a T75 flask one day prior to transfectionsuch that they were 50-70% confluent when used for the experiment. Thehuman NPY Y5 intronless genomic clone HG.PCR15, containing the fulllength open reading frame encoding the receptor, was first linearizedwith Not I and purified using a Wizard PCR Prep kit (Promega). For eachtransfection, 8 μg of linearized DNA were added to 1.25 ml of Optimemculture media (Life Sciences) and 37 μl of Transfectam (Promega) wereadded to 1.25 ml of Optimem. These two solutions were then mixedtogether and added to cells previously washed once with Optimem. Afteran incubation period of 5 hours, the DNA/Transfectam mixture wasremoved, cells were washed with PBS and fed with DMEM supplemented with10% FBS. Cells were left intact for two days, and then switched toselection media (DMEM 10% FBS containing 350 μg/ml of G418) for 5-10days followed by CBLD as described above. The individual clone293.hy5.sb.8 was selected on the basis of its high level of [¹²⁵I]PYYbinding activity, using the intact cell binding protocol from above.

Membrane Preparation

The media was removed from each flask of transfected cells, and thecells were washed twice with 20 ml ice-cold phosphate buffered saline.The cells were scraped from the flask in 5 ml of Tris buffer (20 mMTris-HCl and 5 mM EDTA, pH 7.7), and then transferred to a centrifugetube. Each flask was washed with an additional 5 ml of Tris buffer andcombined in the centrifuge tube. The cells were polytroned for 2×10seconds (12 mm probe, 7000-8000 rpm) and centrifuged 5 minutes (Centra7R, International Equipment Co., Needham Heights, Mass.) at 800 rpm and4° C. The supernatant was then transferred to a clean centrifuge tubeand was centrifuged at 30,000×g for 30 minutes and 4° C. The supernatantwas removed and the pellet was stored at −80° C. Protein concentrationwas measured using the Bio-Rad kit pursuant to the standardmanufacturer's protocol (Biorad Laboratories, Hercules, Calif.),withbovine IgG as the standard.

[¹²⁵I]pYY Binding Assay for NPY Y5 Receptors

The binding assays were performed on GF/C Millipore (Bedford, Mass.)96-well plates pretreated with 0.02% polyethylenimine (PEI) for at least2 hours prior to use. The PEI was aspirated from the plates on a vacuummanifold immediately before the samples were added to the wells. Allpeptides, tissue and radioligand were diluted with binding buffer (25 mMTris, 120 mM NaCl, 5 mM KCl, 1.2 MM KH₂PO₄, 2.5 mM CaCl₂, 1.2 mM MgSO₄,0.1% BSA and 0.5 mg/ml bacitracin, pH 7.4). For competition assays,increasing concentrations of peptide were incubated with [¹²⁵I]PYY andtissue. In a final volume of 200 μl, samples consisted of membraneprotein (ie., 2.5-15 or 10-30 μg membrane protein for rat Y5 or humanY5, respectively); 75-100 pM [¹²⁵I]PYY NEN-DuPont (Boston, Mass.);peptide dilution or binding buffer. Nonspecific binding was defined by 1μM PYY. NPY, PYY, (2-36)NPY, (10-36)NPY, (LP)(3-36)NPY and (32D-Trp)NPYwere synthesized at Bayer Corp. (West Haven, Conn.). All other peptideswere purchased from either Peninsula (Belmont, Calif.) or Bachem(Torrance, Calif.).

For saturation experiments, increasing concentrations of [¹²⁵I]PYY wereincubated with membrane and 1 μM PYY. After a 2 hour incubation at roomtemperature with constant mixing, the samples were aspirated on a vacuummanifold. The wells were washed with three 200 μl aliquots of ice-coldbinding buffer. The individual wells were punched into 12×75 mm plastictubes, and counted on a Wallac (Gaithersburg, Md.) gamma counter.Binding data were analyzed using the nonlinear regression curve-fittingprogram RS/1 (BBN Software Products Corp., Cambridge, Mass.).

Binding Assays for Rat Y2, Y1, and Y4/PPI Receptors

The binding buffer for rat Y2 binding was Krebs/Ringer bicarbonate(Sigma K-4002, S-8875), pH 7.4, containing 0.01% bovine serum albumin(BSA—Sigma A-2153) and 0.005% bacitracin. 0.85-1 μg of protein and 25 pM[¹²⁵I]PYY are added to each well. Nonspecific binding is defined by 1 μMNPY.

The binding buffer for rat Y1 and rat Y4/PPI binding consisted of 137 mMNaCl, 5.4 mM KCl, 0.44 mM KH₂PO₄, 1.26 mM CaCl₂, 0.81 mM MgSO₄, 20 mMHEPES, 1 mM dithiothreitol (DTT), 0.1% bacitracin, 100 mg/l streptomycinsulfate, 1 mg/l aprotinin, 10 mg/ml soybean trypsin inhibitor and 0.3%BSA, pH 7.4. For rat Y1 binding, ˜5-15 μg of protein and 50 pM [¹²⁵I]PYYwere added to each well, and nonspecific binding was defined by 1 μMNPY. For the rat Y4/PP1 binding assay, ˜1-2 μg of protein and 50 pM rat[¹²⁵I]PP (NEN DuPont, Boston, Mass.) were added to each well, and 1 μMrat PP was used to define nonspecific binding.

In Vitro Functional Assay—Measurement of Forskolin-Stimulated AdenylateCyclase

Rat Y5

(Reference: Gordon et al., J. Neurochem. 55, 506, 1990) Suspension cellsstably expressing the Y5 receptor (approximately 400,000 per sample)were resuspended in serun-free DMEM containing 10 mM HEPES (pH 7.4) and1 mM isobutylmethylxanthine (IBMX). 1 uM forskolin was added to thecells. The assay was stopped by transferring the samples into a boilingwater bath for 3 minutes. After a 3 minute centrifugation at 14,000×g,an aliquot of each sample was quantitated for cAMP levels byradioimmunoassay (NEN DuPont, Mass.).

Human Y5

Monolayer cells stably expressing the Y5 receptor were pre-rinsed withWash buffer (pH 7.2: 137 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl₂, 0.5 MMMgCl₂, 6.5 mM Na₂HPO₄, 1.5 mM KH₂PO₄). Cells were then incubated for 10minutes at 37° C. in Assay buffer (pH 7.4: Wash buffer+10 mM HEPES, 10mg/ml BSA, 0.5 mg/ml bacitracin, 0.4 mg/ml soybean trypsin inhibitor).After addition of fresh buffer and 100 μM IBMX, the cells were incubatedfor 10 minutes at 37° C. The reaction was started with the addition ofpeptide and 1-10 μM forskolin. After a 20 minute incubation at 37° C.,the reaction was terminated by discarding the buffer and adding 65%ethanol to each well. The supernatant was then transferred to microfugetubes and the extraction step was repeated once more. After evaporationof the ethanol from the samples, the amount of cAMP was assayed using byradioimmunoassay (MEN DuPont: Boston, Mass.).

In Vivo Pharmacology Procedures

Adult male Wistar rats were surgically implanted with a chronicintracerebral ventricular (ICV) cannula (Plastic Products, Roanoke, Va.)using a stereotaxic instrument. Several days after the surgery, 1-6nmoles of each peptide (or saline) was injected into the lateralventricle of 4-12 rats in a volume of 5-10 μl. The quantity of rodentchow consumed in a 2 hour period was measured.

In Vitro and In Vivo Pharmacology Results

FIG. 1 presents the competition curves of various peptides for [¹²⁵I]PYYbinding to Y5 receptor membranes transiently expressed in COS-7 cells.Each point is the average value of triplicate determinations from arepresentative experiment. IC₅₀ values corresponding to 50% inhibitionof specific binding were determined using nonlinear regression analysis.K_(i) values were calculated from the IC₅₀ values using theCheng-Prusoff correction, such that K_(i)=IC₅₀/(1±(L/K_(d))), where L isthe radioligand concentration and K_(d) is the dissociation constant.The results for transiently expressed Y5 clones are presented in Table1, and Table 2 contains data for stably expressed Y5 clones. TABLE 1 NPYY5 BINDING AFFINITIES (K_(i) ± SEM, nM) RAT Y555 RAT 6B RAT Y861 HUMANPEPTIDE Clone* Clone Clone Clone r/hNPY 0.53 ± 0.06 0.49 ± 0.03 0.50 ±0.06 0.73 ± 0.09 rPYY 1.1 0.48 ± 0.09  1.0 ± 0.13  1.3 ± 0.14 (1.2,0.95) h(LP)PYY 2.5 ± 0.5 0.57 ± 0.01  1.8 ± 0.09 1.7 ± 0.3 r/h(LP)NPY0.96 0.31 0.55 ± 0.11 0.97 ± 0.36 (1.0, 0.92) p(LP)NPY ND 0.64 ± 0.070.47 0.88 ± 0.11 r/h(2-36)NPY 0.81 0.65  1.2 ± 0.07  1.2 ± 0.15 (0.61,1)   p(3-36)NPY 3.6 ± 0.4  1.9 ± 0.27 2.0 10.4 ± 2.0  (1.8, 2.3)r/h(3-36)NPY ND 0.49 2.1  3.8 ± 0.48 (2.7, 1.6) r(3-36)PYY 6.2 ± 1.1 1.4 ± 0.10  4.2 ± 0.47  10 ± 3.4 r/h(10-36)NPY 35 4.9  34 ± 2.8 110(6.0, 3.8) (110, 109) p(13-36)NPY 40 7.7 22 56 ± 7  (38, 41)  (7.9, 7.5)(25, 19) r(13-36)NPY 73  11 ± 1.0 86 ± 19 77 (89, 65) p(18-36)NPY 303194 ± 88  206 ± 61  618 ± 85  r/h(20-36)NPY 636 330 ± 31  587 >1000r/h(22-36)NPY >1000 >1000 >1000 >1000r/h(26-36)NPY >1000 >1000 >1000 >1000 (1-24)NPY ND >1000 >1000 >1000BIBP3226 ND >1000 >1000 >1000 hPP ND ND  4.0 ± 0.29 11  (15, 6.2) rPP ND62 296 ± 47  436 (582, 290)*IC₅₀ values (nM)

TABLE 2 K_(i) Values (nM; Average ± SEM) Peptide 293.hY5.sb.8293S.Y861.2 rPYY 1.3 ± 0.2 0.71 ± 0.1  hPYY 1.1 ± 0.2 1.06 ± 0.2 (3-36)PYY 4.5 ± 0.7 3.6 ± 0.4 (13-36)PYY  24 ± 2.0 29 ± 4  h(LP)PYY 1.3± 0.1 0.76 ± 0.1  r/hNPY 0.79 ± 0.1  0.86 ± 0.07 p(LP)NPY 1.2 ± 0.4 0.67± 0.04 h/r(LP)NPY 0.89 ± 0.1  0.67 ± 0.04 (LP)(3-36)NPY 3.1 ± 0.6 2.9 ±0.9 (2-36)NPY  1.4 ± 0.03 0.83 ± 0.1  h(3-36)NPY 3.5 ± 0.4 1.4 ± 0.4(10-36)NPY  14 ± 2.7  15 ± 4.7 p(13-36)NPY 8.7 ± 1.6 8.8 ± 2.0p(18-36)NPY 144 ± 18  61 ± 13 (20-36)NPY 429 ± 133 108 ± 16 (22-36)NPY >900 >930 (26-36)NPY >900 >930 (1-24)NPY >900 >930(32D-Trp)NPY 7.3 ± 0.8 4.2 ± 1.0 hPP 3.7 ± 1.6 2.5 ± 0.5 rPP 286 ± 77 203 ± 44 K_(i) values for various peptides for [¹²⁵I]PYY binding to thetransiently expressed rat 6B, Y861 and Y555 receptor clones as well asthe human Y5 receptor. The averages±standard error of the mean (SEM)represent values from at least three independent experiments. Twoindependent experiments are represented by the average, followed by theindividual values in parentheses. Remaining values without SEM are froma single experiment. Peptide species inTable 1 (and Table 2, infra) are indicated with the following prefixes:r=rat, h=human, p=porcine, r/h=rat=human. ND=not determined.

The rank order of the affinities of the peptides tested is as follows:NPY˜PYY˜(LP)PYY˜(LP)NPY˜(2-36)NPY˜(3-36)PYY˜(LP)(3-36)NPY˜(3-36)NPY>(32D−Trp)NPY>(10-36)NPY˜(13-36)NPY>(18-36)NPY>(20-36)NPY>>(22-36)NPY,(26-36)NPY

In Table 3, the pharmacological profile of the standard peptides isexpanded for the other cloned NPY receptors to further illustrate thenovel nature of the Y5 receptor pharmacology. In addition, the in vivofeeding response of some of these peptides is listed for comparison. Thedata shown are representative of the average of at least two independentexperiments, as described in the methods. Feeding of rats injected (ICV)with saline was <3 g/2 hours.

Table 4 shows the EC₅₀ values for same standard peptides at the rat andhuman Y5 receptor.

C-terminal fragment (3-36)NPY binds preferentially to Y2 receptors,while (LP)NPY has lower affinity. Conversely, (LP)NPY has high affinityfor the Y1 receptor, while (3-36)NPY and the C-terminal fragments aremuch weaker. When considering the rat Y4/PP1 receptor, rat PP has veryhigh affinity as compared to NPY, PYY, (LP)NPY, and (13-36)NPY. In thein vivo feeding model, (LP)NPY, which has high affinity for Y1 and lowaffinity for Y2, and (3-36)NPY, which has a high affinity for Y2, butnot Y1, all stimulate feeding in rats. Rat PP does not induce muchfeeding when administered to rats. This in vivo profile matches the invitro pharmacological profile outlined in Table 2 for the Y5 receptor.

In addition, while (LP)(3-36)NPY (a custom peptide synthesized at Bayer)has weak affinity for Y1, Y2 and Y4/PP1, it stimulates feeding in rats.Importantly, (LP)(3-36)NPY has high affinity for the Y5 receptor (Table2). These data are further evidence that the Y5 receptor is linked tofeeding. TABLE 3 IC₅₀ VALUE (nM) Rat Y1 Rat Y2 Rat Y4/PP1 Rat Y5 FeedingPEPTIDE (clone) (clone) (clone) (Y861) (g/2 h) r/hNPY 0.13 0.24 >10000.45 >5 rPYY 0.43 0.079 630 0.9 >5 h(Leu³¹Pro³⁴) 0.57 116 ND 2.0 >5 PYYp(Leu³¹Pro³⁴⁾ 0.15 150 4.3 0.63 >5 NPY r/h(2-36)NPY 47 0.50 >1000 1.3 >5p(3-36)NPY 45 0.67 >1000 2.2 >5 r/h(Leu³¹Pro³⁴) 44 154 20 3.4 >5(3-36)NPY hPP 40 >1000 0.065 4.9 >5 (32DTrp)NPY >1000 26 ND 7.0 NDr/h(10-36)NPY 148 0.42 >1000 34 <3 rPP 843 >1000 0.071 325 <3p(18-36)NPY 287 0.34 159 326 <3 (20-36)NPY 435 0.64 ND 638 <3(22-36)NPY >1000 0.89 ND >1000 <3 (26-36)NPY >1000 84 ND >1000 <3(1-24)NPY >1000 >1000 ND >1000 <3The pharmacological profile for the 6B (and Y861 and Y555) receptorclones is distinct from Y1 receptors (wherePYY˜NPY˜(LP)NPY>(3-36)NPY>(13-36)NPY˜(18-36)NPY>(LP)(3-36)NPY), as wellas Y2 receptors (wherePYY˜NPY˜(13-36)NPY˜(18-36)NPY˜(3-36)NPY>>LP)NPY(LP)˜(3-36)NPY). The Y5receptor is also different from the pancreatic polypeptide (PP) receptor(Y4/PP) since [¹²⁵I]PP (rat) does not bind to it.

Although the rank order of affinities is essentially the same whencomparing 6B to Y861 and Y555, subtle differences do exist in the IC₅₀values. It appears that Y861 and Y555 have slightly lower affinities(approximately 2- to 3-fold) for PYY and other PYY analogs, as comparedto 6B. In addition, (10-36)NPY and (13-36) have 2- to 4-fold loweraffinity for Y861 and Y555.

Nonlinear regression analysis of saturation data for the Y5 receptoryielded a K_(d) value of 0.27 nM and a receptor density (B_(max)) ofabout 140 fmol/mg protein in these transiently transfected cells.

FIG. 2 presents the saturation curve for specific binding of [¹²⁵I]PYYto Y5 receptor membranes transiently expressed in COS-7 cells. Membraneswere incubated with concentrations of [¹²⁵]PYY ranging from 0.05 to 5nM, in the presence or absence of 1 μM PYY. Each point represents theaverage value of triplicate determinations at each concentration tested.The inset in FIG. 2 shows the corresponding Rosenthal plot of the dataTABLE 4 EC₅₀ Values, nM (n Value) Peptide 293S.Y861.2 293.hY5.sb.8r/hNPY 6.3 ± 1.9 (3) 0.3 (1) rPYY 6.5 (2) ND r/h(2-36)NPY 21 (2) NDr/h(3-36)NPY ND 6 (1) → r/h(LeuPro)(3-36)NPY 31 ± 39 (3) 23 ± 11 (3)(32D-Trp)NPY 24 (1) 33 (1) hPP 1 (1) 5 (1) rPP 112 (1) >1000 (1)

Example 5 Isolation of Human Y5 Receptor

Isolation of Human Genomic Clone

Polymerase chain reaction (PCR) was used to amplify a 375 base pair (bp)coding region of the rat Y5 cDNA clone. The primers for the PCR were:(+) 5′-TAGGGAACCTGGCCTCCTCC-3′ (SEQ ID NO 5) (nucleotides 487-506), (−)5′-TCAGAGGGCCATGACTCAAC-3′ (SEQ ID NO 6) (nucleotides 843-862).The PCR product was cloned into pCRII vector (Invitrogen) and sequenced.After confirmation by sequencing, the insert was purified from the lowmelting gel and labeled with digoxigenin-11-dUTP using the random primedmethod (Boehringer Mannheim, Indianapolis, Ind.). The labeled probe wasused to screen human genomic library.

1×10⁶ independent recombinants were screened from the library. Filterhybridization was carried out in the hybridization buffer containing6×SSC, 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate (SDS), 3%blocking reagent (Boehringer Mannheim) and 30% formamide at 37° C.overnight. The filters were washed at 37° C. in 0.1×SSC, 0.1% SDS andthe positive clones were identified by CSPD detection kit according tothe manufacturer's protocol (Boehringer Mannheim).

Two positive clones (HG11A and HG19) were isolated from the library. Thepositive clones were subcloned into pBluescript vector (Stratagene). Oneclone, h11a, was analyzed by restriction mapping and plasmid Southernblot. Two EcoRV fragments, 2.4 kb and 0.4 kb, were hybridized by the ratY5 probe. These two DNA fragments were subcloned and sequenced from bothends. DNA sequence analysis was performed using GCG program. The codingregion of the human Y5 genomic clone was identified by DNA sequenceanalysis. This region was amplified by PCR using genomic clone h11A astemplate and subcloned into pcDNA3 expression vector (Invitrogen) forfurther studies. The h11A clone has the nucleic acid coding sequencegiven by SEQ ID NO 5 and the protein that it encodes has the amino acidsequence given by SEQ ID NO 6.

The human Y5 DNA coding region was used to search the sequencesimilarities in the gene bank. The Y5 coding sequence from nucleotide821 to the stop codon at position 1338 is nearly identical, but in anopposite orientation, to part of the human NPY-Y1 gene (Ball et al, J.Biol. Chem. 270, 30102 (1995)). The identical sequence covered the 1Cexon promoter, exon 1C, and part of the intron sequences of the NPY-Y1receptor in an opposite orientation. Compared to the publishednucleotide sequence, the Y5 coding region has a T insertion at position1226 and a TG insertion at positions 1235 and 1236.

1-34. (canceled)
 35. A method of identifying a neuropeptide Y agonist orantagonist comprising contacting a potential agonist or antagonistmolecule with a membrane or membrane preparation comprising a membraneor portion thereof of a cell expressing a nucleic acid having anucleotide sequence substantially the same as SEQ ID NO: 1, SEO ID NO:3, or SEQ ID NO:
 5. 36. A method of identifying a neuropeptide Y agonistor antagonist comprising contacting a potential agonist or antagonistmolecule with a membrane or membrane preparation comprising a membraneor Portion thereof of a cell expressing a nucleic acid having anucleotide sequence substantially the same as SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID NO: 5 wherein the cell is a mammalian cell.
 37. Aneuropeptide Y antagonist comprising a compound identified according toclaim
 36. 38. A method of suppressing the appetite of a mammalcomprising administering to the mammal an appetite suppressing amount ofa neuropeptide Y antagonist according to claim
 37. 39. A method ofsuppressing the appetite of a mammal according to claim 38 wherein theamount of antagonist is from about 0.01 to about 100 mg/kg.
 40. Apharmaceutical composition comprising an effective appetite suppressingamount of an antagonist according to claim 37 together with apharmaceutically acceptable career.
 41. A neuropeptide Y agonistcomprising a compound identified according to claim
 36. 42. A method ofstimulating the appetite of a mammal comprising administering to themammal an appetite stimulating amount of a neuropeptide Y agonistaccording to claim
 41. 43. A method of stimulating the appetite of amammal according to claim 42 wherein the amount of agonist is from about0.01 to about 100 mg/kg.
 44. A pharmaceutical composition comprising aneffective appetite stimulating amount of an agonist according to claim41 together with a pharmaceutically acceptable cartier.
 45. (canceled)